Chapter 8:
Bacteriophage Lytic Enzymes as Antimicrobials

Affiliations: 1: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850;
2: Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;
3: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850;
4: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850

The gram-negative peptidoglycan, which lies subjacent to the outer membrane, is relatively thin and undecorated by surface proteins or carbohydrates. The cell wall binding domain (CBD) epitopes are usually carbohydrates or teichoic acids that are unique to a species, much like a bacterial fingerprint. The most extensively studied lysins in animal models are Cpl-1, an N-acetylmuramidase, and PAL, an N-acetylmuramoyl-Lalanine amidase, both of which are from phages that infect Streptococcus pneumoniae. PlyG demonstrates lytic activity on a variety of Bacillus anthracis strains as well as one Bacillus cereus strain. Importantly, the enzyme was shown to be effective in killing and detecting germinating spores in addition to vegetative cells. The spore coat that normally forms an impenetrable surface for lytic enzymes undergoes an increase in porosity following germination, allowing lysins access to the peptidoglycan. Phage therapy has additional advantages of being self-replicating, has over 100 years of historical use, has obtained some regulatory approval, and can target either gram-positive or gram-negative organisms. Lysin therapy, in contrast, is not self-replicating and at the moment requires additional techniques to show efficacy on gram-negative bacteria. Clearly, both phage therapy and lysin therapy represent reasonable alternatives for management of food-borne pathogens.

Steps to bacterial lysis in phage and lysin therapy. Phage therapy (left) exploits a natural phage lytic cycle, which occurs over 30 min and is divided into three major steps, including the release of new virions into the environment. Subsequent infection of new hosts illustrates the process of self-amplification. The electron micrograph depicts phage particles adhering to the debris of a lysed streptococcal cell. In comparison, lysin therapy and prophylaxis (right) are defined by only two steps, in which purified lysin binds to and rapidly kills, through osmotic lysis, the target pathogen. The electron micrograph depicts a cross section of B. anthracis treated with the purified PlyG lysin showing an externalized cytoplasmic membrane just before lysis. (Reprinted from Fischetti et al. [2006] with permission of the publisher.)

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FIGURE 1

Steps to bacterial lysis in phage and lysin therapy. Phage therapy (left) exploits a natural phage lytic cycle, which occurs over 30 min and is divided into three major steps, including the release of new virions into the environment. Subsequent infection of new hosts illustrates the process of self-amplification. The electron micrograph depicts phage particles adhering to the debris of a lysed streptococcal cell. In comparison, lysin therapy and prophylaxis (right) are defined by only two steps, in which purified lysin binds to and rapidly kills, through osmotic lysis, the target pathogen. The electron micrograph depicts a cross section of B. anthracis treated with the purified PlyG lysin showing an externalized cytoplasmic membrane just before lysis. (Reprinted from Fischetti et al. [2006] with permission of the publisher.)

Bacterial peptidoglycan structure and lysin targets. (1) N-acetylglucosaminidase cleaves the glycan component of the peptidoglycan on the reducing side of GlcNAc. (2) N-acetylmuramidase likewise cleaves the glycan component of the peptidoglycan, but on the reducing side of MurNAc. This activity is commonly referred to as a muramidase or lysozyme. (3) An N-acetylmuramoyl-L-alanine amidase cleaves a critical amide bond between the glycan moiety (MurNAc) and the peptide moiety (L-alanine) of the cell wall. This activity is sometimes referred to generically as an amidase. However, a true endopeptidase, or protease, will also cleave an amide bond, but only if it is between two amino acids. This type of activity may occur in the stem peptide (4) of the cell wall or in an interpeptide bridge (5) connecting two cell wall fragments. CBDs typically bind the peptidoglycan-associated carbohydrate or an epitope directly related to the peptidoglycan structure. Note, the structure of the S. aureus cell wall, which is distinguished by a pentaglycine interpeptide bridge, is shown for illustration purposes. Other bacterial species have interpeptide bridges composed of different amino acids or may lack an interpeptide bridge all together. In these organisms, an mDAP replaces L-Lys and directly crosslinks to the terminal D-Ala of the opposite peptide chain.

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FIGURE 2

Bacterial peptidoglycan structure and lysin targets. (1) N-acetylglucosaminidase cleaves the glycan component of the peptidoglycan on the reducing side of GlcNAc. (2) N-acetylmuramidase likewise cleaves the glycan component of the peptidoglycan, but on the reducing side of MurNAc. This activity is commonly referred to as a muramidase or lysozyme. (3) An N-acetylmuramoyl-L-alanine amidase cleaves a critical amide bond between the glycan moiety (MurNAc) and the peptide moiety (L-alanine) of the cell wall. This activity is sometimes referred to generically as an amidase. However, a true endopeptidase, or protease, will also cleave an amide bond, but only if it is between two amino acids. This type of activity may occur in the stem peptide (4) of the cell wall or in an interpeptide bridge (5) connecting two cell wall fragments. CBDs typically bind the peptidoglycan-associated carbohydrate or an epitope directly related to the peptidoglycan structure. Note, the structure of the S. aureus cell wall, which is distinguished by a pentaglycine interpeptide bridge, is shown for illustration purposes. Other bacterial species have interpeptide bridges composed of different amino acids or may lack an interpeptide bridge all together. In these organisms, an mDAP replaces L-Lys and directly crosslinks to the terminal D-Ala of the opposite peptide chain.

25. Foley,S.,, A.Bruttin, and, H.Brüssow.2000. Widespread distribution of a group I intron and its three deletion derivatives in the lysin gene of Streptococcus thermophilus bacteriophages.J. Virol.74:611–618.